Ecosystem respiration depends strongly on photosynthesis in a temperate heath

We measured net ecosystem CO₂ flux (F _n) and ecosystem respiration (R _E), and estimated gross ecosystem photosynthesis (P _g) by difference, for two years in a temperate heath ecosystem using a chamber method. The exchange rates of carbon were high and of similar magnitude as for productive forest ecosystems with a net ecosystem carbon gain during the second year of 293 ± 11 g C m⁻² year⁻¹ showing that the carbon sink strength of heather-dominated ecosystems may be considerable when C. vulgaris is in the building phase of its life cycle. The estimated gross ecosystem photosynthesis and ecosystem respiration from October to March was 22% and 30% of annual flux, respectively, suggesting that both cold-season carbon gain and loss were important in the annual carbon cycle of the ecosystem. Model fit of R _E of a classic, first-order exponential equation related to temperature (second year; R ² = 0.65) was improved when the P _g rate was incorporated into the model (second year; R ² = 0.79), suggesting that daytime R _E increased with increasing photosynthesis. Furthermore, the temperature sensitivity of R _E decreased from apparent Q ₁₀ values of 3.3 to 3.9 by the classic equation to a more realistic Q ₁₀ of 2.5 by the modified model. The model introduces R _photo, which describes the part of respiration being tightly coupled to the photosynthetic rate. It makes up 5% of the assimilated carbon dioxide flux at 0°C and 35% at 20°C implying a high sensitivity of respiration to photosynthesis during summer. The simple model provides an easily applied, non-intrusive tool for investigating seasonal trends in the relationship between ecosystem carbon sequestration and respiration.     

<Emphasis Type="Italic">Melanopsis</Emphasis> from Al-Qarn,Jordan Valley (Gastropoda: Cerithioidea)

Fossil species ofMelanopsis from a freshwater formation in the Jordan Valley (near Al-Qarn) were investigated and the deposits containing these species are formally described as Al-Qarn Formation. Four species were found:Melanopsis buccinoidea Olivier,M. tchernovi Heller & Sivan,M. costata Olivier andM. aaronsohni Blanckenhorn.Melanopsis costata was represented by two groups, “stepped” and “non-stepped”, the latter differing in its lower figurativity index. Intermediates were found betweenM. buccinoidea andM. tchernovi; they may be hybrids. TheMelanopsis assemblage bridges the faunal gap, in the Jordan Valley, between the 2 Ma lake of ‘Erq el Ahmar on the one hand and the 0.8–1.7 Ma lake of ‘Ubeidiya on the other. This suggests an early Pleistocene age of about 1.8 million years for the Al-Qarn Formation.     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

Export,purification, and activities of affinity tagged <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> levansucrase produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His₆- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His₆-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru. R. Biedendieck and R. Beine contributed equally to this work.     

Clonal expansion of CD8+ effector T cells in childhood tuberculosis

The role of CD8(+) T cells in human tuberculosis (TB) remains elusive. We analyzed the T cell repertoire and phenotype in 1) children with active TB (< or =4 years), 2) healthy latently Mycobacterium tuberculosis-infected children, and 3) noninfected age-matched (tuberculin skin test-negative) controls. Ex vivo phenotyping of T cell subpopulations by flow cytometry revealed a significant increase in the proportion of CD8(+)CD45RO(-)CD62L(-)CD28(-)CD27(-) effector T cells (T(EF)) in the peripheral blood of children with active TB (22.1 vs 9.5% in latently M. tuberculosis-infected children, vs 8.5% in tuberculin skin test-negative controls). Analyses of TCR variable beta-chains revealed markedly skewed repertoires in CD8(+) T(EF) and effector memory T cells. Expansions were restricted to single TCR variable beta-chains in individual donors indicating clonal growth. CDR3 spectratyping and DNA sequencing verified clonal expansion as the cause for CD8(+) effector T cell enrichment in individual TB patients. The most prominent enrichment of highly similar T(EF) clones (>70% of CD8(+) T(EF)) was found in two children with active severe TB. Therefore, clonal expansion of CD8(+) T(EF) occurs in childhood TB with potential impact on course and severity of disease.     

Tumor-associated E-cadherin mutations affect binding to the killer cell lectin-like receptor G1 in humans

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and memory T cells in man and mice. Cadherins were recently identified as ligands for mouse KLRG1 but ligands for human KLRG1 have not yet been defined. In this study, we first demonstrate that human E-cadherin is a ligand for human KLRG1. This finding is remarkable because human and mouse KLRG1 show only an intermediate degree of homology (57% aa identity). In addition, we show that E-cadherin, expressed on K562 target cells, inhibited polyclonal human NK cells. Inhibition of NK cell function was observed consistently in three independent functional assays but the extent of inhibition was modest and required high expression of E-cadherin on target cells. E-cadherin function is often inactivated during development of human carcinomas and splice-site mutations resulting in in-frame loss of exon 8 or 9 occur frequently in diffuse type gastric carcinomas. Our experiments further revealed that interaction of human KLRG1 to E-cadherin was susceptible to these tumor-associated mutations and that KLRG1(+) NK cells were triggered more easily by K562 target cells carrying these mutations in comparison to target cells expressing wild-type E-cadherin. These results also indicate that the E-cadherin binding sites important for homophilic interaction are also involved in KLRG1 binding. Taken together, these data demonstrate that the main adhesion molecule of epithelial tissue, E-cadherin, is involved in regulation of NK cells in both humans and mice.     

Analysis of direct and cross-presentation of antigens in TPPII knockout mice

Tripeptidyl peptidase II (TPPII) is an oligopeptidase forming giant complexes in the cytosol that have high exo-, but also, endoproteolytic activity. Immunohistochemically, the complexes appear as distinct foci in the cytosol. In part controversial biochemical and functional studies have suggested that TPPII contributes, on the one hand, positively to Ag processing by generating epitope carboxyl termini or by trimming epitope precursors, and, on the other, negatively by destroying potentially antigenic peptides. To clarify which of these roles is predominant, we generated and analyzed TPPII-deficient mice. Cell surface levels of MHC class I peptide complexes tended to be increased on most cell types of these mice. Although presentation of three individual epitopes derived from lymphocytic choriomeningitis virus was not elevated on TPPII-/- cells, that of the immunodominant OVA epitope SIINFEKL was significantly enhanced. Consistent with this, degradation of a synthetic peptide corresponding to the OVA epitope and of another corresponding to a precursor thereof, both being proteasomally generated OVA fragments, was delayed in TPPII-deficient cytosolic extracts. In addition, dendritic cell cross-presentation of phagocytosed OVA and of OVA internalized as an immune complex was increased to about the same level as direct presentation of the Ag. The data suggest a moderate, predominantly destructive role of TPPII in class I Ag processing, in line with our finding that TPPII is not induced by IFN-gamma, which up-regulates numerous, predominantly constructive components of the Ag processing and presentation machinery.     

Regulation of carcinogenesis by IL-5 and CCL11: a potential role for eosinophils in tumor immune surveillance

The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor development and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced fibrosarcomas in IL-5 transgenic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-1)-deficient mice that lack a key chemokine that recruits eosinophils into tissues, and the eosinophil-deficient mouse strains, IL-5/CCL11(-/-) and DeltadblGATA. It was found that MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11(-/-) BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinophil-deficient IL-5/CCL11(-/-) and DeltadblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our data support a potential role for the eosinophil as an effector cell in tumor immune surveillance.     

Ligation of B and T lymphocyte attenuator prevents the genesis of experimental cerebral malaria

B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.